Objective To develop a ＲT-PCＲ method for determination of Encephalomyocarditis virus ( EMCV) in Mongolian gerbils and Laboratory mice． Methods The primers were designed and synthesised according to the published EMCV specific sequences of VP1 gene． ＲT - PCＲ method is established，carries on the specificity，sensitivity，stability test． The method is used to detected 62 Mongolian gerbils and 12 mice． Ｒesults The developed ＲT-PCＲ method is good in specificity，ensitivity，stability; and its minimum detection limit using the recombinant plasmid containing EMCV gene as atemplate was 4. 1pg /μL，and the lowest detection virus titer is 10 － 7 ml－ 1． The 62 Mongolian gerbils after ＲT - PCＲdetection were negative; The 12 mice after ＲT -PCＲ detection，there were one EMCV positive，compared with the EMCV in Genebank，The homologies in nucleotide sequence of one positive mice is 85% ; there were 5 mice can be detected EMCV in the body of the 6 mice by artificial infection EMCV． Conclusion The developed ＲT-PCＲ method is good in specificity，ensitivity，stability，can be used in detecting the EMCV in laboratory animal，such as Mongolian gerbils andmice．